New tools for measuring stress response

Oil palm performance under biotic and abiotic stresses can be measured by various means and growth stages. Verdant is working to develop commercial planting materials that can better tolerate drought and Ganoderma, as well as being efficient in fertilizer uptake. This study aims to identify early screening methods for drought tolerance in oil palm which will allow the selection of seedlings and progenies that can be entered into breeding programs and variety production. The trial was arranged according to a split-plot design with three replications. The main plot is watering (normal watering and drought stress) and the sub-plot was Verdant breeding material that consists of 7 progenies. The 4-month-old seedlings were subject to a water deficit treatment (water withheld for five days) followed by measuring seedling responses: leaf temperature, plant height, and number of leaves, which were combined to give a drought score. Significant differences were found for leaf temperature and drought score. Leaf temperature can indicate stress levels of seedlings as it relates to stomatal opening (respiration and transpiration) and hence photosynthetic activity. These methods are being deployed in a phenomics nursery and the data is used to predict subsequent field performance and yield.


Introduction
Oil palm has been grown in almost all main islands of Indonesia (Sumatra, Kalimantan, Sulawesi Moluccas, West Papua, and a small part of Java).Some regions experience several months of water deficit and this has become increasingly common in many oil palm growing areas as a result of climate change.There are reports demonstrating a marked sensitivity of oil palm genotypes to drought.Water deficit impacts inflorescence initiation, sex differentiation, and abortion and decreases oil palm production concerning bunch number [1].Therefore, identifying oil palm genotypes with improved physiological performance under drought conditions is essential for breeding success and in increasing the potential range of environments for oil palm cultivation [2].
Providing materials tolerant to drought stress is a significant component in overcoming this stress.Verdant is developing methods for early screening through seedling trials [3].One method that is being tested involves measuring leaf temperature in response to stress.The method is adapted from banana research where stress is highly correlated with leaf temperature, which is validated by stable isotope discrimination (δ 13 C) [4].Leaf temperature can indicate stress levels of seedlings as it relates to stomatal opening (respiration and transpiration) and hence also photosynthetic activity.Cooler leaf temperatures indicate healthy plants with continuing vegetative growth.Here we describe work in setting up leaf temperature assessments of seedlings subject to drought stress.The experiments involve testing a small but diverse range of germplasm in a drought house facility (shaded and rain sheltered) with the aim to develop a standard simple and early test for drought stress.
Earlier work on imaging for stress response showed variation between control and drought-treated seedlings (Figure 1).By using vegetation indices such as Normalised Difference Vegetation Index (NDVI) the plots subjected to drought showed both different physical appearance and spectral values.The control plots tended to have higher NDVI values than plots subjected to drought, indicating more damage due to drought (decrease in vegetation indices).This work showed that imaging could be a valuable tool to monitor and assess stress response and we wished to extend this to thermal imaging.

Facilities, materials and treatments
Studies were conducted using a drought house at Verdant Bioscience Plantation Science Centre.The trial design was set up on the basis of a split-plot design with four replicates and four seedlings per plot.Table 1 shows the seven progenies tested.The two main plot treatments were: 1.Normal watering.[5] 2. Drought stress -water withheld for 5 days.The sublots were Verdant breeding material that consist of 7 progenies.

Drought assessment method
Germinated seeds were planted in a small polybag (18 cm x 21 cm) and grown for four months in an open nursery.Care was taken not to disturb roots on transferring seedlings to the drought house at four months old (to prevent roots from growing into the ground and being severed on transfer).The treatments (see above) were applied for five days, and data was collected for leaf temperature and performance (plant height and number of leaves).Leaf temperature was captured using a handheld Seek Shot PRO Thermal Imaging Camera for the third youngest leaf from the growing point.Leaf symptoms (Figure 2) were given a drought score as follows: Score 1 : All leaves green and turgid.Score 2 : More than 75% of leaves are green but shrunk or less turgid.

Leaf temperature and growth measurement
Thermal camera images were used to monitor and observe oil palm seedling responses to drought stress.Figure 3 shows images of seedling progenies after five days of drought treatment.The spectrum reflects the temperature of the captured image from the lowest to the highest temperature.T was used to estimate the leaf temperature in normal and drought-stressed plants.The temperature scale is shown in the lower part of the pictures, from black to white in the range from 32°C to 44°C.Data on all variables were statistically analyzed by a split-plot two-way analysis of variance (ANOVA).

Seedling performance during treatment
The means of the progenies under the two treatments are shown in Table 2, and the differences between treatments are in Table 3.The means of progenies in the two treatments are presented in Table 2, and the differences are in Table 3.The mean leaf temperature differences ranged from 4.2 in Progeny 2 to 3.0 in Progeny 6, as shown in Figure 4. Concerning progeny, significant differences were present for plant height (p<0.001) but not for leaf temperature or number of leaves.Drought stress can affect seedling performance.Water availability has a significant effect on leaf temperature (p<0.001) and plant height (p<0.005).Plants that were subjected to drought stress had higher leaf temperatures compared to the control.Control seedlings were also taller compared to stressed seedlings.However, the number of leaves showed no significant differences between the two treatments.Significant differences (p<0.05) can be seen for plant height and, as expected, seedlings under normal watering had better growth performance compared to drought, as shown in Tables 2 and 3 as well as Figure 5. Drought can inhibit shoot growth as there is a lower level of inputs for photosynthesis [7].The biggest difference in height is seen for Progeny 5 (2.8 cm), followed by Progeny 4 (1.6 cm); the most negligible difference is 0.  Seedlings of differing genotypes show differing responses to drought stress in terms of leaf temperature, drought score, plant growth, and number of leaves.Plant height and number of leaves did not show differences and these parameters were not useful in this study.The slight difference in leaves number is probably because leaves appear at the rate of only one per month until seedlings are 6month-old [8].The visual parameters are also subjective and not easily defined or quantified.Drought treatment also had significant effects on leaf drought scores (p=00.1).Control seedlings consistently had a healthy drought score (Score 1), whereas, under drought stress, the drought score varied from 2.4 to 3.8.Significant differences existed between progenies for drought response (p<0.001).The results of the multiple-range test are reported in Figure 6.Progeny   highest drought score followed by progeny 6 and 4. Progeny 5 showed the lowest drought score and was thus classified as being the most susceptible to drought from the perspectiveof this assessment method.

Correlation
Correlation analysis was used to compare the different measurements (Table 4).The highest correlation was the positive value reported for leaf temperature with a drought score (0.85).This correlation implies that leaf temperature increases reflect the increase in the drought score (the score based on visual observation.)However, with the low number of progenies, the other parameters do not show significant correlations with the number of leaves or plant height.Leaf symptoms have been used traditionally for thousands of years as an indication of plants lacking water, and so the relationship with leaf temperature measurements means it is a promising tool to assess stress in plants.

Conclusions
The thermal camera was found to be useful in measuring drought stress in oil palm seedlings.It should be noted that leaf temperature is influenced by the surrounding ambient environment, and this can distort leaf temperature measurements, thus reducing this environmental effect is important and can be achieved by making observations at a specific time [9].In addition, the thermal camera can capture and record the temperature quickly and accurately, so data in the nursery can be collected much faster than traditional methods.Parallel measurements of light intensity, ambient temperature, and humidity can be complementary data to improve accuracy in data collection.The choice of leaf sample is also essential avoiding dead plant/tissue.It is suggested that in carrying out drought treatment the critical point of drought stress is best measured when leaves show a score of 3.Otherwise, they may have already died, in any case, will not recover even though rewatered.Theseresults are preliminary but do show the potential for using rapid measurements of leaf temperature to assess plant water stress status and can be used to identify progenies more tolerant to drought.

Figure 1 .
Figure 1.Spectral camera imaging of drought stress in nursery.

Score 3 :
More than 50% of leaves (especially the older ones) are pale green, showing partial dehydration.The bottom leaf turns yellow to brown.Score 4 : More than 50% of leaves (especially the older ones) are turning brown, showing severe wilting or folding, and partially dehydrated.Score 5 : Leaf completely brown and desiccated.

Figure 2 .
Figure 2. Leaf score from 1 to 5 from left to right.

Figure 3 .
Figure 3. Seedling performance after 5 days drought treatment of different progenies: (a) drought stress (b) normal watering, colour spectrum shows the temperature for the leaf colour images.

Figure 4 .
Figure 4. Graph of leaf temperatures in the control and under drought stress at five days of treatment.

Figure 5 .
Figure 5. Graph of mean plant height in control and drought stress at five days of treatment.

Figure 6 .
Figure 6.Leaf drought score of seven oil palm progenies 5 days after treatment; bars with different letters indicate significant differences (P<0.001) according to Least Significant Difference test.
[1] Suharyanti N A, Mizuno K and Sodri A 2020 The effect of water deficit on inflorescence period at palm oil productivity on peatland E3S Web of Conferences vol 211 (EDP Sciences) [2] Silva P A, Cosme V S, Rodrigues K C B, Detmann K S C, Leão F M, Cunha R L, Festucci Buselli R A, DaMatta F M and Pinheiro H A 2017 Drought tolerance in two oil palm hybrids as related to adjustments in carbon metabolism and vegetative growth Acta Physiol Plant 39

Table 1 . Progeny details Progeny number Breeding background* Cross type
[6]r more information on the genetic diversity of the progenies see Nur et al.[6]

Table 2 .
Progeny means for control and stress treatment for drought score, leaf temperature, plant height, and number of leaves after 5 days of treatment.

Table 3 .
Differences in progeny means for control and stress treatment (Diff.) for drought score, leaf temperature, plant height, and number of leaves after five days of treatment.

Table 4 .
Correlation coefficients between the parameters, drought score, leaf temperature, number of leaves and plant height.